Effects of focused ultrasound in a “clean” mouse model of ultrasonic neuromodulation

Summary Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.


INTRODUCTION
1][22][23][24][25][26][27] A recent detailed biophysical study in cultured cortical neurons found that the mechanical effects of FUS excite neurons via specific mechanosensitive ion channels. 28However, in live rodents, FUS can activate peripheral auditory pathways and cause off-target activation throughout the brain, including both ipsilateral and contralateral regions, regardless of the specific brain targets being stimulated, presenting a persistent challenge in UNM experiments in rodents. 29,302][33] To eliminate these confounds in rodents, using deaf animal models is an effective approach.However, surgical or chemical methods of deafening can be invasive or cause systemic toxicity, which limits the potential for fully awake and long-term chronic experiments. 29,30,34A recent UNM study used genetically deaf knockout mice with deficits in the inner hair cells, 35 but their congenital deafness may impair brain plasticity and cortical development, potentially limiting their utility in experiments involving sensory processing, learning, and other cognitive functions. 36,37Therefore, it remains challenging to non-surgically deafen adult normal hearing mice without compromising their capability for neural recording and behavioral experiments.
Here we address this challenge by developing a new ''clean'' mouse model for ultrasound (US) research and characterizing the safe parameter range for future UNM and sonogenetics studies.In contrast to a knockout mouse model, the double transgenic mouse model (Pou4f3 +/DTR 3 Thy1-GCaMP6s) does not inactivate specific genes expressed in the brain.Instead, it has human diphtheria toxin receptor (DTR) placed downstream of the Pou4f3 promoter, sensitizing hair cells to diphtheria toxin (DT), which allows us to use DT to rapidly, non-invasively, and selectively ablate all the hair cells without causing systemic toxicity to the mice at any point in life. 38These mice will maintain normal hearing after birth, which promotes normal brain development and potentially makes them appropriate candidates for conducting high-level behavior experiments.
Additionally, other genetically deaf knockout mouse models did not express fluorescence in the brain. 35In contrast, our mice express calcium indicator GCaMP6s in the brain, enabling us to simultaneously read neuronal activity across different cortical regions in awake, deafened mice during UNM.
Using this new mouse model, we show that FUS-elicited auditory confounds and off-target brain activation can be effectively reduced or eliminated up to certain pressures in fully awake, deafened mice.As the pressure was increased, the fluorescence signal at the FUS focus gradually decreased due to focal heating and the fluorophore's thermal dependence.Additionally, high-pressure FUS elicited bilateral off-target brain activation from non-auditory brain regions, possibly due to the activation of non-auditory peripheral sensory receptors.When the pressure exceeded 1,600 kPa, we observed strong calcium responses originating from the FUS focus and subsequently propagating throughout the ipsilateral hemisphere.This strong depolarization was associated with brain damage, as confirmed by histology, indicating that high-pressure FUS causes damage-evoked seizure-like spreading depolarization.These findings provide key insights for future UNM and sonogenetics studies.First, they offer an effective deaf mouse model where the sensory confounds of UNM can be eliminated up to a certain pressure.Second, they delineate an FUS parameter space where non-auditory confounds and tissue damage can be minimized.Third, with thermal fluorescence dimming, they provide a convenient approach to visualize the focus of FUS in the cortex.

RESULTS
Diphtheria toxin deafens double transgenic Pou4f3 +/DTR x Thy1-GCaMP6s mice Transgenic Pou4f3 +/DTR (Pou) mice were bred with Thy1-GCaMP6s (Thy1) mice to produce double transgenic Pou4f3 +/DTR 3 Thy1-GCaMP6s (PouThy1) mice, expressing the heterozygous human DTR from the endogenous Pou4f3 locus, 39 and the fluorescent calcium indicator GCaMP6s. 40The PouThy1 mice maintain normal hearing and balance until being treated with DT (Figure 1A), which ablates all their inner and outer hair cells. 39The deafness of the treated mice can be examined by imaging cortical auditory responses through a clear skull (Figures 1B and 1C).In awake mice, we compared the auditory responses to audible broadband noise and visual responses to light flashes among three groups, which were Thy1 mice treated with DT (Thy1-DT), PouThy1 mice treated with saline (PouThy1-saline), and PouThy1 mice treated with DT (PouThy1-DT).Audible sound (90 dB sound pressure level) activated the auditory cortex and other cortical regions in Thy1-DT and PouThy1-saline groups while no such activation pattern was observed in the PouThy1-DT group (Figures 1D-1F), suggesting DT can effectively deafen PouThy1 mice.In contrast, light flashes (80 ms in duration) evoked similar calcium responses in the visual cortex among all the three groups, suggesting that DT does not damage non-auditory cortex (Figure 1G).

Deafening reduces or eliminates off-target widespread cortical activation to US
We further investigated if the PouThy1-DT mouse model could eliminate the auditory confounds and the off-target widespread cortical activation reported by previous UNM studies. 29,30Our in vivo UNM setup comprised a wide-field camera and a single element transducer angled to the brain for simultaneous FUS stimulation and calcium imaging. 30We used this setup to avoid potentially artifactual mechanical interactions between recording electrodes and FUS, and because wide-field imaging enables us to capture larger areas of the brain, facilitating the assessment of off-target and localized effects caused by FUS.The tip of the transducer (fundamental frequency at 270 kHz and third harmonic at 916 kHz) was manually aligned to the stimulation target, which is À0.5 mm anterior and 2.5 mm lateral of Lambda (Figure 2A).Two previous studies, which primarily examined pulsed parameters in deafened rodents, suggested that these parameters do not cause direct brain activation. 29,30Therefore, in the current study, we primarily focused on a continuous FUS waveform with a pulse duration (PD) of 500 ms, which has been directly demonstrated to effectively activate cultured cortical neurons via specific mechanosensitive calcium channels in an environment free from auditory confounds. 282][43][44] In addition to continuous stimulation, our study examines representative pulsed stimulation parameters.
As expected, when using the 270 kHz FUS, we were able to observe strong auditory confounds and off-target brain activation in normal hearing mice (Thy1-DT).In contrast, in deafened mice (PouThy1-DT), the widespread cortical responses were mostly eliminated when using low peak negative pressure FUS (%500 kPa).Unexpectedly, we still clearly observed bilateral off-target brain activation from non-auditory regions when increasing the pressure to 900 kPa (Figures 2B and 2F-2H).To examine if this activation was directly elicited by targeted FUS, we compared the ipsilateral and contralateral responses of the visual cortex to FUS, but we did not find that the ipsilateral focus had stronger responses than its contralateral counterpart (Figures 2D and 2E).This suggests that the activation is not likely to be caused directly by local FUS stimulation of the targeted visual cortex.
To assess the impact of focal zone size, we stimulated with the third harmonic of our transducer, at 916 kHz, with a lateral full width at half maximum (FWHM) pressure profile of 1.4 mm so that the focus can be confined within one hemisphere (Figures 3 and S2).As expected, in Figure 2. Deafening reduces off-target responses to 270 kHz ultrasound (A) Illustration of simultaneous ultrasonic neuromodulation (UNM) and wide-field cortical imaging.The ultrasound (US) transducer and the imaging equipment were both angled at 45 from parallel to allow optical access to the focus.The transducer was immersed in a cone filled with degassed water.The cone was then coupled to the skull with degassed US gel, which was flattened with a glass plate.The temperature of the gel mound was regulated at approximately 35 C using bilateral copper bars via heat conduction.The other ends of the bars were sealed in 3D printed tubes and submerged in circulating warm water to maintain a constant temperature.(B) Representative examples of cortical activation map from one normal hearing mouse (Thy1-DT) and one deafened mouse (PouDTR-DT) to sham, US (270 kHz center frequency, 500 ms PD, pressure at 100, 500, and 900 kPa), and light flashes.The US target zone is shown as a black circle.The boundary maps are the same as in Figure 1.In the sham trials, no stimulus was presented or applied.(C) Visual responses to light flashes of normal hearing and deafened mice, and the normalized (detailed in STAR Methods) peak dF/F of the two groups (n = 6 mice for each group, unpaired t test, two-tailed, p = 0.2656).The onset time of stimulation is shown as a vertical blue line.(D and E) Cortical responses at ipsilateral (ipsi) US focus and its contralateral (contra) counterpart to US at 500 kPa (D) and 900 kPa (E) and light flashes.The normalized peak dF/F are compared (n = 6 animals, unpaired t test, two-tailed, p = 0.9755 and 0.8999 for D and E, respectively).(F-H) Auditory, visual, somatosensory, and motor responses to sham and US at different pressures.Normalized peak dF/F are compared between the two groups (n = 6 animals for each group, unpaired t test, two-tailed, *p < 0.05, **p < 0.01, ns is not significant).Mean trace is solid and SEM is shaded.Bar graph values represent mean G SEM. normal hearing mice, the application of FUS to the visual cortex resulted in widespread cortical activation at pressure as low as 100 kPa.In contrast, in fully deafened mice, the off-target brain activation was eliminated at pressures up to 900 kPa.Taken together, these results reinforce the importance of using fully deafened mice for UNM experiments because FUS can produce strong auditory and widespread brain activation in normal hearing mice at pressure as low as 100 kPa.In addition, they reveal the additional concern of non-auditory side effects when using low-frequency transducers with large focal zones relative to brain size.

US reduces focal fluorescence in deafened mice via a thermal mechanism
In the clear skull preparation, we noticed a decrease in local calcium indicator fluorescence following high pressure (900 kPa, 500 ms PD) FUS stimulation (Figure S2), which is in line with the temperature-dependent fluorescence dips reported in another recent UNM study. 42To better characterize the relationship between continuous FUS heating and fluorescence without the potentially distorting effects of the skull, we replaced bilateral skull regions with TPX, an acoustically and optically transparent polymethylpentene plastic, 0.125 mm in thickness. 45The windows covered +3 to À4 mm anteroposterior (AP), +1 to +5 mm mediolateral (ML) for each hemisphere (Figure 4A).The large windows can accommodate the full 916 kHz FUS focus and allow imaging of major portions of the bilateral motor, somatosensory, and visual cortices.
When increasing the peak positive pressure from 100 kPa to 1,000 kPa in increments of 300 kPa, we observed the decrease of focal fluorescence following the onset of FUS, which then gradually returned to the baseline (Figures 4B and 4C).We measured the temperature change at the brain surface under the TPX window with a miniaturized thermistor during UNM in separate animals (Figures 4D and S5) and calculated our in vivo preparation having a temperature dependence of fluorescence of approximately À0.85%/ C (Figure 4E), relatively  consistent with Estrada's result of À1.9 G 0.7%/ C in brain slices. 42The difference may be due to heating of the TPX window material or the thermistor used for our measurement, leading to an overestimation of temperature changes.Further investigations should account for factors like the skull and/or cranial window during thermal modeling, 11,46,47 and use miniaturized devices for temperature measurement.
To study the effects of temperature on cellular GCaMP fluorescence, we heated a population of GCaMP6f-expressing human HEK293 cells using a qPCR machine from 37 C to 42 C in 1-degree increments and simultaneously measured cellular fluorescence.We found a linear decrease in fluorescence intensity when the temperature increased (Figure 4F), suggesting that the observed US-induced fluorescence dimming in vivo could, at least in part, be attributed to the US-induced elevation in temperature.

High-pressure US elicits focal, spreading depolarization with underlying tissue damage
Having failed to obtain evidence of direct brain activation in awake deafened mice with FUS at pressures up to 1,300 kPa and a long interstimulus interval (ISI) of 155 s (Figure S3), we further tested if pressures of 1,600 kPa and above (in increments of 400 kPa at 916 kHz) could elicit stronger calcium responses, potentially overcoming the negative fluorescence dip caused by heating.At this higher pressure, we observed a very strong calcium signal at the focus ($200% peak dF/F, Figure 5), which was $20-fold larger than that elicited by sensory stimuli (e.g., Figure 1).Originating at the FUS focus, this excitation propagated throughout the ipsilateral hemisphere over approximately 1 min (Figure 5).This spatiotemporal pattern is similar to that elicited by focal cortical seizures. 48,49On the contralateral side, there was a relatively weaker (about 15% peak dF/F) brain activation having similar duration with the ipsilateral response.We also found that the strong focal calcium signal was not readily repeatable in the same animal.As shown in an example (Figure 5C), when performing five consecutive FUS trials (Stim 1 to 5) at an interval of 10 min in one experiment, only the first and third trials resulted in signal, indicating potential tissue damage.To determine effects on the tissue, we perfused three animals 24 h after they received a single pulse of US stimulation (2,000 kPa, 500 ms PD, 916 kHz) and performed H&E staining, with two animals showing clear damage in the area of stimulation (Figure 5E).The combined live imaging and histological evidence suggests that the strong propagating calcium signal was due tofocal neuronal damage leading to spreading activation over the ipsilateral cortex, rather than direct non-damaging US stimulation.

Low-pressure pulsed US does not elicit localized calcium signals
After comprehensively characterizing responses to continuous FUS, we also tested pulsed stimulation parameters (Table 1) informed by UNM studies that have reported that low-intensity pulsed FUS can elicit electrophysiological activity at the FUS focus. 2,50To keep the FUS focus on the ipsilateral target and minimize somatosensory effects, we used 916 kHz FUS instead of 250-500 kHz FUS used in their studies.We did not observe any activation in our deafened mice with TPX window (Figure 6), even with 3-to 4-fold higher pressure than used in previous reports.In contrast, we observed strong bilateral brain activation in normal hearing mice using the same parameters (Figure S4), suggesting that auditory confounds may contribute to the widespread brain activation elicited by pulsed FUS.

DISCUSSION
This study presents a new conditionally deafened mouse model for studying the effects of FUS on neural activity in vivo and detailed characterization of the cortex-wide calcium dynamics of deafened mice in response to continuous FUS with 500 ms PD, at pressures ranging from 100 kPa to 2,400 kPa.In clear skull preparation, by eliminating the auditory confounds, we observed a significant reduction in off-target brain activation with 270 kHz FUS at pressures up to 500 kPa, and complete elimination of off-target activation with 916 kHz FUS at pressures up to 900 kPa.][53] Using the TPX window preparation with 916 kHz continuous FUS for 500 ms PD, we observed pressure-dependent off-target and localized effects.After eliminating the auditory confounds, we observed localized temperature-dependent fluorescence dimming starting from pressure at 400 kPa due to fluorophore heating (Figure 4), which is consistent with a previous report. 42When we increased the pressure to 1,000 and 1,300 kPa, we occasionally observed bilateral off-target neural activation at the visual and somatosensory cortices in conjunction with dimming of ipsilateral fluorescence (Figure S3).When we further increased the pressure to 1,600 kPa or above, we observed strong focal depolarization with spreading to ipsilateral cortex, accompanied by damage to brain tissue and contralateral off-target sensory activation.
To understand the off-target residual sensory effects in deafened mice induced by FUS, we created a finite element model (FEM) of a mouse 54 and used it to investigate the response of mouse models to transcranial FUS applied to the visual cortex (Figure 7).We found that the induced wave pattern is complex and has both localized and delocalized components, suggesting that peripheral skin receptors and photoreceptors may experience displacements and stresses at levels that could result in the activation of ascending somatosensory and visual pathways, as reported in some peripheral UNM studies. 43,52,53,55We also computed the off-target stresses at a small region on the mouse neck, where we observed that the shear stresses (i.e., von Mises stresses), unlike pressures, exhibit a strong frequency dependence in their response (Figures 7B and 7C).These modeling results, together with our experimental data, establish a range of pressure-dependent localized and off-target effects of FUS UNM (Figure 7D).Furthermore, as auditory confounds have been reported both with perpendicular and angled transducers, 29,30,35,50 future studies should investigate the detailed differences in sound and shear wave propagation patterns in the mouse skull and their implications for UNM.
We found no evidence that FUS evokes direct calcium responses in the live brain (Figures 3, 4, and S3) at parameters that were able to do so in cultured neurons (Figure S6). 28This discrepancy may be due to biophysical differences, as the brain may experience different mechanical and acoustic conditions than cultured neurons.Moreover, the expression levels of the US-sensitive and amplifier channels identified in cultured embryonic neurons (TRPP1/2, TRPC1, and TRPM4) may be different in the adult mouse brain.Future studies may explore celltype responses to FUS in fully deafened animals to better understand this discrepancy.
Previous studies investigated the correlation between FUS parameters and motor readouts of brain activation (i.e., electromyography [EMG] signals) in normal hearing, lightly anesthetized animals. 3,4,41In our study, we used fully deafened, awake mice and relied on cortical calcium responses as direct indicators of brain activation.Due to these differences, the findings concerning effective parameters in previous studies may not directly map onto ours.
However, our results do not conclusively demonstrate that FUS is unable to produce direct brain activation.For example, although a comprehensive set of continuous FUS parameters had been tested, including 270 kHz and 916 kHz center frequency, other frequencies and PDs could yield different results.Furthermore, moderate-pressure FUS may have generated weak activation masked by temperaturedependent fluorescence reduction.Similarly, high-pressure FUS may have produced brain activation that was overwhelmed by the intense seizure-like depolarization attributed to cellular damage.Finally, there might be subcortical activation and finer cortical activation that cannot be observed by wide-field calcium imaging. 30,56][59] Our study employed a conditionally deafened animal model, which exhibits nearly complete hair cell loss in the cochleae and utricles, 39 making it an optimal model for studying the use of treatments such as deep brain stimulation, transcranial magnetic stimulation, and transcranial direct current stimulation, for hearing and vestibular disorders.8][59][60] However, it is crucial to use models like our conditionally deafened mice because our previous and current experimental and modeling results have shown that transducers at center frequencies lower or around 1 MHz could elicit strong auditory confounds and off-target brain activation. 29,30,54Moreover, researchers should also pay attention to somatosensory and visual confounds, as high-pressure FUS may still activate ascending non-auditory pathways in deafened animals.To distinguish brain activation from disruptive depolarization, it is necessary to perform histology and confirm that the FUS parameters leading to brain activation do not cause any brain damage.In conclusion, UNM holds great potential in treating various brain disorders, 61,62 and it is always advisable to stay cautious by imaging whole cortical or brain activity and performing control experiments to ensure that the observed brain activation is localized and not confounded.Further studies using optical readouts in conditionally deafened animal models will help validate parameters and mechanisms for translational UNM and sonogenetics in humans and help define a parameter range considered safe from thermal and other potentially damaging effects.

Limitations of the study
This study explored a limited range of cortically targeted FUS parameters using wide-field calcium imaging in awake, deafened Pou4f3 +/DTR 3 Thy1-GCaMP6s mice.While we did not observe direct neural stimulation, we do not rule out that such stimulation may be possible and The pulse patterns of parameter set #1 overlapped with that being used in Tufail's study 2 while the pulse patterns of parameter set #2 overlapped with that being used in Yu's study. 50These two studies are representative of many others in the field. 19Note that we used 916 kHz FUS with smaller focus to minimize the offtarget effects induced using a low-center frequency transducer.(D) US can elicit auditory confounds at low pressure.In deafened mice, the auditory confounds can be greatly reduced or eliminated.However, the thermal confounds at the focus, other sensory confounds, and destructive depolarization will be observed as the pressure of the US increases.

Figure 1 .
Figure 1.Cortical responses to sham, audible sounds, and light flashes (A) Diagram of protocol for mouse deafening.Two injections of diphtheria toxin (DT) or saline were spaced two days apart.Wide-field imaging experiments were performed at least two weeks after the first injection to wait for the ablation of hair cells in the cochlear and utricles.(B) Illustration of wide-field calcium imaging setup.A speaker was positioned in the front of the mouse.Two LEDs were positioned to the right and left eyes, respectively.(C) Illustration of the top view of the cortex from Allen Mouse Brain Common Coordinate Framework (CCFv3).The visual, auditory, somatosensory, and motor cortices were indexed with numbers.(D and E) Representative examples of cortical activation map to sham, light flashes to both eyes, audible broadband noises to both ears.Two animals are presented for each group.In the sham trials, no stimulus was presented or applied.(F) Auditory responses to audible sounds and the peak dF/F of the Thy1-DT, PouThy1-saline, and PouThy1-DT groups (n = 10 mice for each group, one-way ANOVA ****p < 0.0001, Tukey's post comparison).The onset time of stimulation is shown as a vertical blue line.(G) Visual responses to light flashes and peak dF/F of visual responses of the Thy1-DT, PouThy1-saline, and PouThy1-DT groups (n = 10 mice for each group, oneway ANOVA p = 0.1050, ns is not significant).Mean trace is solid and SEM is shaded.Bar graph values represent mean G SEM.

Figure 3 .
Figure 3. Deafening eliminates off-target responses to 916 kHz ultrasound (A) Representative examples of cortical activation map from one normal hearing mouse (Thy1-DT) and one deafened mouse (PouDTR-DT) to sham, US (916 kHz, 500 ms PD, pressure at 100, 300, and 500 kPa), and light flashes.The US target zone is shown as a black circle.In the sham trials, no stimulus was presented or applied.(B) Visual responses to light flashes of normal hearing and deafened mice, and the normalized peak dF/F of the two groups (n = 6 animals for each group, unpaired t test, two-tailed, p = 0.8618).The onset time of stimulation is shown as a vertical blue line.(C) Cortical responses at US focus to sham, US at different pressures, and light flashes, and the normalized peak dF/F (n = 6 animals, one-way ANOVA ****p < 0.0001, Tukey's post comparison) of the deafened mice.(D-F) Auditory, visual, and somatosensory and motor responses to sham and US at different pressures.Normalized peak dF/F are compared between the two groups (n = 6 mice for each group, unpaired t test, two-tailed, *p < 0.05, **p < 0.01, ns is not significant).Mean trace is solid and SEM is shaded.Bar graph values represent mean G SEM.

Figure 4 .
Figure 4. Temperature dependence of fluorescence in in vivo deafened mouse brain and in vitro human cells (A and B) Two representative examples of cortical activation map in response to sham, US at different pressures (916 kHz, 500 ms PD, pressure at 100, 400, 700, 1,000 kPa), and light flashes.A raw image of a TPX window mouse is shown in A. The length and posterior width of each window are approximately 7 and 4 mm, respectively, allowing for complete acoustic access to the brain for the 916 kHz US.The US target zone is shown as a black circle.The white/black polygonal dots represent the boundaries of the TPX windows.In the sham trials, no stimulus was presented or applied.(C) Focal calcium responses to sham and US at different pressures (i.e., 100, 400, 700, 1,000 kPa) across four animals.Mean trace is solid and SEM is shaded.(D) In vivo measurement of the brain temperature increases during UNM.Intensities of 0.33, 5.33, 16.33, 33.33 W/cm 2 correspond to pressures of 100, 400, 700, 1,000 kPa, respectively.The measured temperature increase linearly correlates with the US intensity (R 2 = 0.997).(E) Fluorescence change is plotted against measured temperature increase with a slope of À0.85%/ C for in vivo brain.(F) Fluorescence change is plotted against measured temperature increase with a slope of À0.49% C for in vitro HEK293T cells.

Figure 5 .
Figure 5. Ultrasound produces localized and hemispherically spreading disruptive depolarization (A and B) Two representative examples of cortical activation maps of localized depolarization by US in deafened animals.The depolarization started at the focus and then propagated over the ipsilateral hemisphere.The right panels are the dF/F of the ipsilateral focus (red curve) and its contralateral counterpart (black curve).The ipsilateral dF/F is approximately 20-folds of the contralateral dF/F.The US target zone is shown as a yellow/black circle.The black polygonal dots represent the boundaries of the TPX windows.The onset time of stimulation is shown as a vertical dot line in the right plots.

Figure 5 .
Figure 5. Continued (C) The dF/F at the focus in response to five consecutive US stimulation (2,000 kPa, 500 ms PD) with an interval of 10 min.The zoomed curves are shown below.Only trial #1 and #3 elicited depolarization.(D) The peak dF/F is 2.31 G 0.20, onset time is 3.87 G 3.07, and peak time is 30.5 G 6.35 s, respectively (n = 3 animals).Bar graph values represent mean G SEM. (E) Examples of H&E staining at the sonication site suggest that high-pressure US (2,000 kPa, 500 ms PD) induced brain damage (white regions with low cell density, as pointed by black arrows) at the focus.

Figure 7 .
Figure 7. Localized and non-localized effects and potential mechanisms of in vivo UNM in mice (A) Result of finite element method (FEM) model of a mouse, where an FUS with peak amplitude of 1,000 kPa and frequency of 916 kHz is applied as a normal pressure introduced as a Neumann boundary condition.The results depict the pressure contours after 200 ms of explicit dynamics simulations.Note the pressure is widely distributed on the head.(Band C) Time history of off-target pressures and von Mises stress at a region on the face, obtained from two separate FEM simulations with identical FUS peak amplitudes of 1,000 kPa but distinct frequencies.(D) US can elicit auditory confounds at low pressure.In deafened mice, the auditory confounds can be greatly reduced or eliminated.However, the thermal confounds at the focus, other sensory confounds, and destructive depolarization will be observed as the pressure of the US increases.

Table 1 .
Pulsed ultrasound parameters investigated in deafened mice